A Novel Multiplexed Immunoassay Identifies CEA, IL-8 and Prolactin As

Mahboob et al. Clinical Proteomics (2015) 12:10 DOI 10.1186/s12014-015-9081-x CLINICAL PROTEOMICS

RESEARCH Open Access A novel multiplexed immunoassay identifies CEA, IL-8 and prolactin as prospective markers for Dukes’ stages A-D colorectal cancers Sadia Mahboob1, Seong Beom Ahn1, Harish R Cheruku1, David Cantor1, Emma Rennel3, Simon Fredriksson3, Gabriella Edfeldt3, Edmond J Breen4, Alamgir Khan4, Abidali Mohamedali2, Md Golam Muktadir5, Shoba Ranganathan2, Sock-Hwee Tan1, Edouard Nice6 and Mark S Baker1*

Abstract Background: Current methods widely deployed for colorectal cancers (CRC) screening lack the necessary sensitivity and specificity required for population-based early disease detection. Cancer-specific protein biomarkers are thought to be produced either by the tumor itself or other tissues in response to the presence of cancers or associated conditions. Equally, known examples of cancer protein biomarkers (e.g., PSA, CA125, CA19-9, CEA, AFP) are frequently found in plasma at very low concentration (pg/mL-ng/mL). New sensitive and specific assays are therefore urgently required to detect the disease at an early stage when prognosis is good following surgical resection. This study was designed to meet the longstanding unmet clinical need for earlier CRC detection by measuring plasma candidate biomarkers of cancer onset and progression in a clinical stage-specific manner. EDTA plasma samples (1 μL) obtained from 75 patients with Dukes’ staged CRC or unaffected controls (age and sex matched with stringent inclusion/exclusion criteria) were assayed for expression of 92 human proteins employing the Proseek® Multiplex Oncology I proximity extension assay. An identical set of plasma samples were analyzed utilizing the Bio-Plex Pro™ human cytokine 27-plex immunoassay. Results: Similar quantitative expression patterns for 13 plasma antigens common to both platforms endorsed the potential efficacy of Proseek as an immune-based multiplex assay for proteomic biomarker research. Proseek found that expression of Carcinoembryonic Antigen (CEA), IL-8 and prolactin are significantly correlated with CRC stage. Conclusions: CEA, IL-8 and prolactin expression were found to identify between control (unaffected), non-malignant (Dukes’ A + B) and malignant (Dukes’ C + D) stages. Keywords: Multiplex immunoassay, Plasma biomarker, Colorectal cancer

Background Australian Clinico-pathological staging (ACPS) system CRC is the third most commonly diagnosed cancer and Dukes’ staging system [2,3]). Patient prognosis in- worldwide with over 694,000 deaths (8.5% of all cancer versely correlates with tumour stage at the time of diagno- deaths) in 2012, with Australia and New Zealand having sis [4,5]. Once metastases becomes clinically observable, the highest incidence rates (44.8 and 32.2 per 100,000 in prognosis is extremely poor with survival often measured men and women respectively) [1]. in months [6]. Currently, we are unable to detect patients Various staging systems have been developed to de- with clinically silent metastases, possibly linked to poor scribe the progression of the disease based on the size, outcome. Despite the availability of numerous screening location and spread of the tumour to distant organs strategies, aggressive surgical therapy and extensive re- (e.g., Tumour-Node-Metastasis (TNM) staging systems, search on the molecular basis of CRC, early detection of the disease remains problematic. Population-based CRC * Correspondence: [email protected] screening programmes can reduce morbidity and mortal- 1Australian School of Advanced Medicine, Faculty of Medicine and Human Sciences, Macquarie University, Rm1, Level 1, 75 Talavera Road, Sydney, NSW ity through the early identification of surgically-treatable 2109, Australia disease. However, there is currently a gap in translational Full list of author information is available at the end of the article

© 2015 Mahboob et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mahboob et al. Clinical Proteomics (2015) 12:10 Page 2 of 12

research between identification of potential new bio- evaluated simultaneously using the Proseek assay. The markers and development of Food and Drug Association levels of 8 oncoproteins (CEA, IL-8, prolactin, amphiregu- (FDA) approved diagnostic tests [7]. Most diagnostic tests lin, PDGF-BB, IL-6, CXCL11 and CXCL5) differed signifi- available to date are based on a single protein biomarker cantly between various individual CRC stages (Table 1). [8]. This concept is hazardous in the clinical setting as bio- Additional file 1: Tables S3 and S4 list the complete logical systems are interdependent and highly complex statistical analyses on this data. Twelve (12) of the target with inherent false positives subject to the genomic in- biomarkers were found to have expression levels below stability of cancers [9]. It is now widely accepted that the Proseek LOD. panels of biomarkers will be required to achieve the in- Specifically, CEA was found to be the overexpressed creased sensitivity and specificity necessary for population- protein measured in stage D when compared with any based screening [10]. The use of a pan-cellular field such other CRC stage (i.e., either Dukes’ A, B or C) and/or as proteomics could help identify protein expression pro- healthy unaffected controls (for all stage D comparisons files associated with CRC progression that may prove to Ps were ≤ 0.0001). In addition, CEA was also overex- be more reliable than single biomarker based assessment. pressed in stage C when compared with stage A (P = Simultaneous assessment using a multiple biomarker 0.0076) and/or healthy controls (P = 0.0304). Previous strategy necessitates the development of multiplex high- studies have also shown elevated CEA expression in throughput technologies with sufficient sensitivity and Dukes’ stage C and D CRC [15-21]. specificity to detect CRC early [9]. Multiplexing or simul- Differences in IL-8 expression were observed in stage A taneous quantitation of several biomarkers in plasma can to D comparisons (P = 2.96E-05) and stage D to healthy indicate the protein expression profiles involved in tumour control comparisons (P = 1.23E-06). Interestingly, levels of formation, progression and metastasis. Under carefully prolactin were elevated in Dukes’ stage C compared with controlled experimental conditions, multiplexed assays stage D (P = 1.24E-05) and healthy controls (P = 2.89E-05). can identify many (96) low abundance candidate proteins Prolactin levels were found to consistently increase as dis- using minimal sample volumes (1 μl) [11]. An example of ease progressed from controls through CRC Dukes’ stages this technology is the proximity extension assay (PEA) A-C (Figure 1, P5). Amphiregulin was overexpressed in which has recently been developed by Olink Biosciences stage D when compared with stages A (P = 8.96E-07) and from Uppsala, Sweden [12]. This study was designed to meet a longstanding clin- Table 1 Tukey-honest significant differences post-hoc ical need for earlier CRC detection by identifying plasma test for Proseek data [Stage specific (A-D)] and healthy biomarkers of CRC onset and progression using the unaffected control (group E) Proseek® Multiplex Oncology kit I (Proseek assay). In de- Candidate Comparison Up/Down of Adjusted Previous studies tail, Proseek assay employs PEA technology to quantitate Biomarker expression p-value referring CRC 92 potential oncoproteins using only 1 μL of human associations plasma [12], where samples are treated with matched CEA D/A ↑ 0[15-21] antibody pairs that are tagged with DNA reporter mole- D/E ↑ 1.70E-12 cules. Once the antibodies are bound to their respective D/B ↑ 4.13E-12 antigen the corresponding DNA tails form an amplicon D/C ↑ 0.0001 that can be quantified by high-throughput real time PCR C/A ↑ 0.0076 which generates a measurable fluorescent signal that dir- C/E ↑ 0.0304 ectly correlates with abundance [13]. This PEA-based approach provides a platform for accurate quantification IL-8 D/E ↑ 1.23E-06 [44,47] of multiple (96) low abundance oncoproteins from bio- D/A ↑ 2.96E-05 logical samples. Here, we aimed to validate PEA results Prolactin C/D ↑ 1.24E-05 [49-51] with an existing benchmark multiplexed technology, C/E ↑ 2.89E-05 namely the Bio-Plex Pro™ human cytokine 27-plex kit Amphiregulin D/A ↑ 8.95E-07 [54] [14] (Bio-Plex), which is a bead-based multiplex im- D/C ↑ 1.46E-06 munoassay, measures the concentrations of 27 cytokines, chemokines or growth factors. PDGF-BB D/A ↑ 3.02E-05 [22,55] IL-6 B/A ↑ 0.0024 [43,56] Results B/E ↑ 0.0124 Proseek® multiplex oncology I assay CXCL11 D/C ↑ 0.0155 [57] The expression levels of 92 potential protein biomarkers D/A ↑ 0.0387 (Additional file 1: Table S3) in each of the 75 plasma CXCL5 D/A ↑ 0.0424 [58-61] samples from CRC patients and healthy controls were Mahboob et al. Clinical Proteomics (2015) 12:10 Page 3 of 12

Figure 1 Panels (P) (A, B, C, D & E) representing protein abundance data for CEA (A), IL-8 (B) prolactin (C), amphiregulin (D) and PDGF-BB (E) in individual Dukes’ stage A, B, C & D with E controls; pooled group (defined control, non-malignant, malignant groups). Values were determined by Proseek PEA using CRC patient EDTA plasmas and was based on z-scores (log2 scale).

C(P = 1.46E-06), while PDGF-BB was elevated in stage D This study showed expression of three biomarker compared with stage A (P = 3.02E-05). It was also noted oncoproteins (CEA, IL-8 and prolactin) were altered that IL-6 showed a higher expression in stage B when when pooled CRC groups (i.e., control, non-malignant, compared to stage A and healthy unaffected controls malignant) were considered. (P ≤ 0.0024). Chemokine (C-X-C motif) ligand CXCL11 Comparison between pooled controls, non-malignant and CXCL5 had a higher expression at stage D when com- and malignant groups with individually staged patients in- pared with stage A (P =0.0155).Itwasinterestingtonote dicated CEA and IL-8 were both considerably upregulated that some of the previously reported biomarker oncopro- in malignant compared to healthy controls. Additionally, teins (e.g., IL-4, CAIX, TNF-α, MCP-1, GM-CSF, VEGF, as expected [20] CEA was overexpressed in comparisons TIE2, IL17, IL-6, IFNG) did not display differential expres- between non-malignant and malignant groups (Q-value = sion between Dukes’ CRC stages (P ≈ 1.0) [22-32]. 0). In contrast, prolactin demonstrated a noticeable Dukes’ To determine whether changes were observed when stage-dependant increase in expression until metastasis CRC data were pooled into control (group E), non- occurredbeyondlymphnodes(i.e.,iselevatedupto malignant (stages A + B combined) or malignant groups stage C). Once metastasized to distal organs (Dukes’ stage (stages C + D combined), a Tukey honest significant differ- D), plasma prolactin expression levels returned to approxi- ences post-hoc ANOVA (Type II) test was performed and mately normal control (group E) levels. Additionally, an Q values calculated, where Q values are a measure of stat- increase was found between control and non-malignant istical significance in terms of false discovery rate (Table 2). pooled groups for prolactin values. Mahboob et al. Clinical Proteomics (2015) 12:10 Page 4 of 12

Table 2 Q values of significantly altered potential biomarker proteins between pooled CRC groups Candidate Biomarker Comparison Difference Lower CI Upper CI Q-value CEA Malignant/Non-malignant 1.97 1.08 2.85 0 CEA Malignant/Healthy 2.11 1.02 3.19 0 IL.8 Malignant/Healthy 1.22 0.14 2.31 0 Prolactin Non-malignant/Healthy 1.1 0.02 2.19 0.04

The Proseek multiplexed assay data strongly suggests from healthy to malignant and may be useful to distin- the combined use of CEA, IL-8 and prolactin expression guish healthy patients from malignant cancers. A converse as potential combined diagnostic indicators of Dukes’ trend pattern was observed for both trends E (amphiregu- stage with their abundance positively correlating with lin, CA19, caspase 3, CD30, CD40, CD69, EGF, GDF15, Il- metastatic progression. However, as target proteins dis- 6, Il-7, Il-8, PDGF-BB) and F (CEA) with strong steady play differences in expression trends, SOMs were used increases observed during progression. Data shown in to cluster and visualise pooled data into one of six dis- trend F demonstrates the power CEA has over all other cernible expression trends (Figure 2). protein biomarkers examined in the Proseek panel for dis- SOMs assembled the data into the six trends (Figure 2), tinguishing malignant from either non-malignant CRC where median values were used as they are less susceptible and/or healthy patients. to variation [33]. The largest differences were observed between either the non-malignant or the malignant patient Bio-Plex Pro™ human cytokine 27-plex immunoassay groups against control patients’ plasmas. SOM trend A Where possible the significant differences observed using biomarkers decrease between controls and non-malignant the Proseek assay were reproduced/validated using an plasmas but increase again to similar levels when malig- established antibody-based multiplexed detection system, nant plasmas are compared to controls. All trend B bio- namely the Bio-plex Pro™ human cytokine 27-plex im- markers show no major change irrespective of stage. In an munoassay. The Dukes’ CRC stage specific analyses identi- opposite manner to trend A, trend D biomarkers increase fied 6 target proteins (IFN-G, IL-4, IL-8, MCP-1, MIP-1 in non-malignant but decrease again in malignancy. Trend and PDGF-BB) that were each significantly elevated C biomarkers steadily decreased biomarker expression in stage D plasmas compared with healthy unaffected

Figure 2 SOMs trends (A-F) of control, non-malignant and malignant CRC groups normalised median Proseek biomarker protein expression scores, showing these can be clustered into six distinct trends. Mahboob et al. Clinical Proteomics (2015) 12:10 Page 5 of 12

controls (P < 0.05) (Table 3). Additional file 1: Tables S5 followed by a small decrease when the malignant group and S6 summarize the statistical analyses conducted on was compared to the non-malignant group. Collectively, the Bio-plex data. these observations may hold some prognostic value for In detail, significant differences in IL-8 expression were evaluation of CRC over healthy controls using Bio-Plex observed in stage A, B and D when compared to healthy analysis of plasma G-CSF, IFN-G, IL-4, IL-6, IL-8, IL-9, controls (P = 6.00E-05, 6.00E-03 and 2.00E-03 respect- MCP-1, MIP-1B and PDGF-BB in a clinical setting. ively). Additionally, PDGF-BB was significantly elevated in stage D compared with healthy controls or stage A (Ps= Comparison of Proseek with Bio-plex data 2.00E-07 and 4.00E-03 respectively). It was also noted that The 13 common proteins that were available across both monocyte chemotactic protein-1/C-C motif chemokine 2 the Proseek and Bio-Plex platforms were evaluated by (CCL2) showed a significantly higher expression in stage pairing and subsequently analysing the combined data D when compared to healthy unaffected controls (P = by Spearman’s rank-order correlation (Figure 4). The X- 5.00E-04). Furthermore, IFNG, IL-4 and monocyte che- Y comparisons for the Bio-Plex (X) and Proseek (Y) data motactic protein-1/C-C motif chemokine 3 (CCL3) also for these common 13 plasma proteins are provided in exhibited significant overexpression in stage D when com- Additional file 1: Table S7. pared to healthy controls (P ≤ 0.05). When comparing the two multiplexed immunoassay Comparison between pooled control, non-malignant and platforms, there were significant differences between out- malignant groups with individually staged patients indi- puts. For a number of proteins (GM-CSF, IL-2, IL-4 and cated that four proteins (i.e., G-CSF, IL-4, IL-8 and MCP-1) TNF), the correlation was highly skewed as the target bio- were more highly expressed between non-malignant and marker fell below LOD in one or both of the platforms. healthy cohorts whilst nine proteins (G-CSF, IFN-G, IL-4, However, IL-8 and MCP-1 scatter plots suggested there IL-6, IL-8, IL-9, MCP-1, MIP-1B and PDGF-BB) were was a positive correlation between data derived from both higher in the metastatic group compared to healthy co- platforms. The scatter plot data for plasma IL-7, IFN-g, horts (Additional file 1: Table S6). SOM analyses of the IL-6, VEGF-A levels also suggested moderate correlation. same data was performed (Figure 3). However, the correlation between Proseek and Bio-Plex Analysis of the SOM data highlights six different analyses for PDGF-BB was particularly strong (p and q trends (A-F) of patient plasma cytokine response to values = 0) (Additional file 1: Table S7). CRC progression. Trend A shows a variable response, In summary, out of the 13 proteins common to both whilst trend B shows an increase in cytokine/chemokine immunoassay platforms, two proteins were identified expression in both non-malignant and malignant CRC whose plasma abundance were differentially correlated groups above healthy controls. Trend C also displays in- with Dukes’ CRC clinical stage (IL-8 between samples creased expression in both cancer groups compared to for Dukes’ D/healthy controls and PDGF-BB between healthy controls with additional slight increases in ma- Dukes’ D/A). The remaining eleven proteins did not lignant above non-malignant groups. The tendency for show major expression difference (P > 0.05) across all increased expression as cancers progress was more pro- comparisons made between Dukes’ stage A-D CRC and nounced in trends D and E, whilst in trend F the in- themselves or healthy controls. For those proteins only crease in non-malignant over control groups was available for assay in a single kit, a small number of proteins showed significantly higher expression profiles Table 3 Tukey-honest significant differences post-hoc test (8 proteins by Proseek CEA, IL-8, prolactin, amphiregu- for Bio-Plex data [Stage specific (A-D)] and healthy lin, PDGF-BB, IL-6, CXCL11 & CXCL5 and 6 proteins unaffected control (group E) by Bio-Plex: G-CSF, IFN-G, IL-4, IL-8, MCP-1, MIP-1 & Candidate Comparison Adjusted p-value Previous studies PDGF-BB). Biomarker referencing CRC association Collectively, these differentially expressed plasma pro- ’ IL-8 D/E 6.00E-05 [44,47] teins epitomise potential Dukes stage- and progression- specific CRC biomarkers. The significant differentially A/E 6.00E-03 expressed proteins identified in this study should now be B/E 2.00E-03 progressed to a much larger double-blind, multi-centre PDGF-BB D/E 2.00E-07 [22,55] biomarker trial for further validation of their potential as D/A 4.00E-03 “combinatorial signatures” of Dukes’ stage-specific CRC. CCL2 D/E 5.00E-04 [61-63] IFNG D/E 0.002 [63] Discussion To the best of our knowledge, this study is one of the IL-4 D/E 0.004 [64] first to simultaneously evaluate two independent multi- CCL3 D/E 0.004 [65] plexed biomarker detection technologies using the same Mahboob et al. Clinical Proteomics (2015) 12:10 Page 6 of 12

Figure 3 SOMs trends (A-F) of control, non-malignant and malignant CRC groups normalised median Bio-Plex biomarker protein expression scores showing these can clustered into six distinct patterns/trends. clinical CRC plasma samples. Choi et al., undertook an cancer research. It is now realised that the limiting factor EDTA (Ethylenediaminetetraacetic acid) plasma proteomic in multiplexed immunoassays is antibody cross-reactivity study using 2DE/MALDI MS combined with Milliplex that typically limits the degree of assay multiplexing [36]. MAP Human 26 Plex Cytokine/Chemokine Kit to investi- Problems with cross-reactivity are virtually eliminated in gate CRC biomarker signatures [34], but have been heavily PEA assays since only matched DNA reporter pairs (i.e., criticized for lacking comparison with age- and other mirroring the presence of two distinct epitopes on the criteria-matched healthy controls [35]. In contrast, the protein) are amplified at the real-time PCR step [12]. current study reports the expression profile of 92 potential Additionally, the small sample volume required (1 μL) to oncoproteins biomarkers from patient EDTA plasmas simultaneously quantitatively assay 92 oncoproteins in a across the four Dukes’ CRC stages combined with an age-, multiplexed format is significantly lower than required for sex-, smoking- and other contraindication-matched healthy alternative platforms (e.g., Luminex-based platforms). This group using the recently developed Olink PEA technology. is important as clinical samples are frequently volume lim- The combined data emanating from the use of this novel ited, particularly when multiple assays (e.g., biomarker multiplexed platform combined with stringent clinical ex- “signature” panels) are required or only limited plasma clusion and inclusion criteria, sample processing and ana- sources (e.g. young children/neonates) are available. As lysis indicates some proteins may be representative of the PEA technology has only recently been commercial- different aspects of progression through Dukes’ staging and ized, it was important to validate the platform against an potentially reflect real differences in CRC biology in vivo existing benchmark technology (e.g., Bio-Plex multiplex during these stages. As such, this study proposes a po- immunoassay) which has FDA approval [37]. The expres- tential biomarker “signature” of clinical relevance that sion profiles of 13 common oncoproteins were compared could be utilised to evaluate Dukes’ CRC stage and between both platforms. Nine of those common proteins progression. showed reasonable correlation between platforms, thereby The Proseek® Multiplex Oncology I assay is a high supporting/validating the potential use of the Proseek throughput, high sensitivity, specific assay developed for assay for cancer biomarker research (Figure 4). Mahboob et al. Clinical Proteomics (2015) 12:10 Page 7 of 12

Figure 4 Spearman correlation scatter plots for 13 individual common biomarkers (PDGF-BB, IL-8, MCP1, IL-6, IL-7, VEGFA, IFNG, IL-2, GM-CSF, IL-4, IL-1ra, IL-12, and TNF) common to the Proseek and Bio-Plex multiplexed immunoassays.

Typically immunoassays involve conformational/shape Investigation of individual CRC stage differences in recognition. As such, the knowledge of epitope location the expression of 92 oncoproteins assessed by the Pro- or structure is crucial for designing targeted, multiple seek assay, identified eight oncoproteins that appear to epitope-based immune assays that can tag multiple se- be differentially expressed in plasma as a result of CRC quences for antigen detection. Inadequate mapping can progression (Table 1). CEA, IL-8 and prolactin demon- yield false positive or negative results. The Proseek assay strated the greatest potential use as diagnostic CRC uses two proximal epitopes to recognize a single antigen Dukes’ stage-specific biomarkers (Figure 1). It was inter- thereby reducing false positive rates which then fall esting to note that except for CEA, none of the other below the LOD (GM-CSF, IL2, IL4 & TNF; Figure 4). significantly expressed oncoproteins matched with the However, this may not be a general phenomenon. Pro- list of serological CRC indicators identified in a similar seek technology uses unique sequence-based tagging of study utilizing a 74-plex PEA platform [39]. In that every antibody in the assay. By contrast, although the study, Thorsen et al., investigated 74 different protein Bio-Plex utilizes a dual antibody system, lack of se- biomarkers and found carcinoembryonic antigen (CEA), quence tagging can create a higher possibility of cross- transferrin receptor-1 (TFRC), macrophage migration reactivity and non-specificity [38]. It should be noted inhibitory factor (MIF), osteopontin (OPN/SPP1) and that both platforms employ automated software and cali- cancer antigen 242 (CA242) as CRC discriminators. bration updates to reduce the need for operator input. CEA, TFRC and CA242 were suggested to be early stage Mahboob et al. Clinical Proteomics (2015) 12:10 Page 8 of 12

CRC indicators. Intriguingly, Choi et al., identified 24 Conclusions significantly elevated proteins in CRC, of which IL-8, New, improved and volume-sparing plasma biomarkers/ TNF-alpha, and IP-10 (interferon gamma-induced biomarker signature panels are urgently required for protein) were elevated in the CRC group relative to the cancer screening and surveillance using minimally inva- adenoma group [34]. Surprisingly, CEA was not a se- sive techniques. Multiplexing represents a powerful plat- lected as a potential oncoprotein in the Milliplex MAP form for the qualitative and quantitative assessment of Human 26 Plex Cytokine/Chemokine Kit used in that cancer biomarker signatures, giving the opportunity for study. sensitive and specific detection of multiple oncoproteins CEA is currently employed as a routine marker for CRC in plasma samples, providing a suite of biomarkers with prognosis, disease-free survival and therapeutic response potential for use as stage-specific indicators of CRC [40] and as an independent predictor of patients at higher progression. risk of CRC recurrence and/or metastases during postop- The emerging PEA technology has received increasing erative follow-up [41]. Our study supports aspects of this interest considering the large number of target bio- contention - namely that high plasma CEA expression sig- markers that can be measured quantitatively with the nificantly correlates with the presence of metastatic CRC, advantage of minimum cross-reactivity over benchmark though (as previously proposed) it was not found to be an multiplexing platforms. This study has identified eight effective plasma biomarker of very early stage disease proteins (CEA, IL-8, prolactin, amphiregulin, PDGF-BB, (Dukes’ A). Another differentially expressed protein (IL-8) IL-6, CXCL11 and CXCL5) whose expression trends are is recognized as a pro-inflammatory cytokine and an im- of great interest for developing a “biosignatures” of CRC portant chemoattractant factor for leukocytes. IL-8 has progression that could potentially be translated into a been reported to contribute to cancer progression through diagnostic/prognostic. Finally, we recognized three pro- potential motility-stimulating, mitogenic and angiogenic spective novel markers of CRC progression (CEA, IL-8 functions [29]. It has been previously demonstrated that and prolactin) that hold potential to be utilised in IL-8 is elevated at both the mRNA and plasma protein clinical oncology, as they significantly increase with levels and in CRC tumour tissues compared to adjacent CRC progression and correlate with Dukes’ stage. We normal colonic mucosa [42-44]. IL-8 is a soluble mediator recognize the importance of performing further multi- released by tumor cells that functions within the tumor centred large study analysis of marker combinations and microenvironment [45]. A number of studies have con- in developing new algorithms that confirm improved firmed the effects of elevated IL-8 on signalling that pro- performance of CEA, perhapsby addition of IL-8 and/or motes the angiogenic response and that eventually leads prolactin. As such, we highly recommend the use of to infiltration of neutrophils to the tumor site [46]. IL-8 these oncoproteins in patient screening as well as for expression in tumour tissues significantly correlates with further investigation as potential CRC plasma bio- tumour size, depth of infiltration, liver metastasis and markers in large multicentric multisample controlled tumour stage [24,47]. The present study also confirms that CRC study cohorts. plasma IL-8 concentration significantly discriminates Dukes’ stage D (those with metastatic disease) from either Materials & methods healthy controls or Dukes’ stage A patients. Patient plasma samples A number of studies have found that prolactin ac- Clinically staged CRC (Dukes’ A, B, C, D) and control tively participates in tumorigenesis and that it is over- EDTA-plasma samples were obtained from 75 patients. expressed in several cancer cell lines including those Patients were Dukes’ staged CRC (15 patients each stage derived from reproductive and non-reproductive tissues A-D) or apparently healthy disease unaffected controls [48]. Hence scientists have been interested in develop- at Victoria Cancer Biobank (n = 15, called group E sub- ing therapies for controlling tumor growth through sequently). Samples were stringently age and sex suppression of prolactin production [48]. Elevated matched with strict inclusion/exclusion criteria applied serum prolactin has been shown to correlate with CRC to minimize variation within the study population. In malignancy [49,50] and is observed in many CRC cell detail, the study population was a mixture (50:50) of lines and tumour specimens [51,52]. Our data strongly females/males, aged between 50 and 80 for each CRC suggests that plasma prolactin significantly correlates stage and for the healthy unaffected controls. Samples with CRC tumour progression through Dukes’ stage A, were collected from CRC patients diagnosed with non- B and C, being continuously upregulated until distal malignant/malignant tumors, before they underwent any metastasis occurs. Our data encourage further studies treatment and surgery for CRC. The control or un- on larger clinical cohorts to evaluate the plasma expres- affected plasma samples were collected from 15 individ- sion of prolactin during CRC progression as a stage- uals who were aged-matched to the clinical CRC plasma specific biomarker. and had no apparent evidence of diseases (i.e., with no Mahboob et al. Clinical Proteomics (2015) 12:10 Page 9 of 12

evidence of inflammation or metastatic conditions, no assay. Data was normalized and analysed using GenEx previous history of tumor, cancer or major therapy. Clin- software (MultiD, Gothenburg, Sweden). All statistical ical details about CRC patients are provided in Additional analyses (dynamic principal component analysis and one file 1: Table S8). way ANOVA) were performed on normalized data. For each biomarker, the limit of detection (LOD) was defined Plasma handling conditions as the mean of negative control plus 3 standard deviations The samples were collected in 2 EDTA tubes (9 ml of the 38 negative controls. each), centrifuged at room temperature (RT) at 1,200 g for 10 mins and plasma fractions transferred to a single Bio-plex Pro™ human cytokine 27-plex immunoassay 10 ml tube. The combined plasma fractions were centri- Bio-Plex assay (Bio-Rad, CA, USA, Cat No: M500KCA- fuged at RT 1,800 g for 10 mins and aliquoted into 8 × FoY) was utilised according to manufacturer’s instruc- 250 μl tubes that were stored and frozen at -80oC. The tions and also as reported earlier [23] to measure the entire process was completed within 2 h of plasma col- concentrations of 27 target proteins (Additional file 1: lection to meet the VCB protocols required for prote- Table S2) using identical CRC plasma samples as used omic experiments. in the Proseek assay. Samples were prepared using a robotic liquid handling workstation (epMotion 5075, Proseek® multiplex oncology I assay Eppendorf, Germany) and incubated with antibody- Proseek assays were performed to evaluate the ex- coupled beads for 60 min followed by incubation with a pression of a panel of potential biomarkers within the detection antibody for 30 min. The conjugates were then plasma samples (n = 75) in a 96-well plate. This assay incubated with streptavidin for 10 min, washed using measured 92 potential protein biomarkers (Additional the Bio-Plex Pro II wash station (Bio-Rad, CA, USA), re- file 1: Table S1) and 4 internal controls generating 9,216 suspended and vortexed prior to fluorescent measure- data points per run. The investigation was performed ac- ment on a Bio-Plex® 200 system (Bio-Rad, Hercules, cording to manufacturer’s instructions (www.olink.com) CA). Data were acquired with the Bio-Plex Systems 100 with minimal changes. The complete experiment was (Bio-Rad, CA, USA), analysed and standard curves (Log conducted on a single 96 well plate to minimize vari- (x) – Linear(y)) generated using the Bio-Rad Bio-Plex ation, with one EDTA plasma sample from each group Manager v6.0 software [53]. (number 15) examined in duplicate. Briefly, 1 μl of each sample or negative control was incubated with the con- Statistical analysis and correlation jugated antibodies at 4°C overnight. The next day, the Biomarker expression values were analysed using the stat- extension mixture was added and the products were ex- istical analytical package RStudio version 0.97.551 across tended and pre-amplified using PCR (ABI 2720 Thermal each of stages A-D CRC and E (apparently healthy un- cycler, Life Technologies). The detection reagent and a affected controls). In addition, to determine if protein ex- fraction (2.8 μl) of the extended and pre-amplified prod- pression could distinguish non-malignant from malignant uct were mixed and loaded into an oil-loaded Fluidigm CRC, individually staged data were pooled into three Gene Expression 96 × 96 Dynamic arrays (Fluidigm Cor- groups, namely controls (Group E; n = 15), non-malignant poration) on one side and the Primer plate with specific CRC (Groups/stage A and B; n = 30) and malignant CRC primers on the other side of the chip. The chip was (Groups/stage C and D; n = 30). This subsequent analysis primed using Fluidigm IFC controller HX (Fluidigm was performed to determine if significant protein expres- Corporation) and afterwards loaded into a Fluidigm Bio- sion differences were found between non-malignant and marker system (Fluidigm Corporation). malignant CRC or when each group was compared to Raw data was annotated using Real Time PCR soft- control patients. A two-way ANOVA using type II sums ware (Fluidigm Corporation). The Proseek assay gener- of squares was used for analysis as the control group was ated Cq values that represent the cycle values in the now smaller than the combined non-metastatic and meta- PCR amplification where the signal is above background. static groups implementing an R notation given as: Log2 This is calculated on a log 2 scale as PCR amplification (Response) ~ Group*Proteins + Sample, where response is is increased by 2 fold up during each cycle. To even out the protein expression value, Group is a factor with 3 variation between and within runs, data was normalized levels (control, non-metastatic and metastatic), Proteins is using the extension control and a background value. The a factor with 92 levels for Proseek data set and 27 levels data used for further statistical analysis were expressed in for the Bio-Plex data set and Sample is a factor with 15 Normalized Protein eXpression (NPX) units on log2 scale, levels. Sample represents the plasma samples from CRC where a high value corresponded to high protein concen- patient and unaffected controls that were included in the tration. The limit of detection (mean negative control plus analysis to allow for any individual differences between 3 × standard deviation) was determined for each protein patients that might mask the difference in expression Mahboob et al. Clinical Proteomics (2015) 12:10 Page 10 of 12

levels between CRC stages. P value < 0.05 was consid- Macquarie University BioFocus and Biomolecular Frontiers Research Centres. ered significant. We sincerely thank the Victorian Cancer Biobank for the collection, supply and provision of inclusion and exclusion criteria matching Dukes’-staged To identify which proteins were differentially expressed CRC EDTA plasmas and for all appropriate clinical data. We also thank for between collated CRC groups (control, non-malignant and statistician Mr Apurv Goel (APAF) for his assistance with data analysis and malignant), a Tukey honest significant differences post- statistics. Some of the research described herein was facilitated by access to the Australian Proteome Analysis Facility (APAF) established under the hoc test was also performed with respect to the interaction Australian Government’s National Collaborative Research Infrastructure between Protein and Group factors. To discover which Strategy (NCRIS). The study was conducted under Macquarie University proteins exhibited similar expression profiles during CRC Human Ethics Committee approval (Approval No 5201200702). progression, self-organizing maps (SOM) were employed Author details to present a discrete representation of the input space of 1Australian School of Advanced Medicine, Faculty of Medicine and Human the protein expression values. Raw data was transformed Sciences, Macquarie University, Rm1, Level 1, 75 Talavera Road, Sydney, NSW 2109, Australia. 2Department of Chemistry and Biomolecular Sciences, Faculty by taking the median expression value for the patient data of Science, Macquarie University, Sydney, NSW 2109, Australia. 3Olink for each protein within each group. This data was log2 Bioscience, Dag Hammarskjölds Väg, 54A, 75183 Uppsala, Sweden. 4Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW transformed and then each group was normalized by set- 5 ’ 2109, Australia. School of Science and Health, University of Western Sydney, ting each groups mean value to zero. Expression data was NSW, Australia. 6Department of Biochemistry and Molecular Biology, Monash clustered into 6 groups using the SOM and displayed as a University, Clayton Campus, Melbourne, VIC 3800, Australia. plot of the normalized median expressions for each pro- Received: 29 October 2014 Accepted: 4 March 2015 tein with respect to its group values. To investigate the complementarity of Proseek and Bio-Plex data sets, the expression of 13 proteins that are References common to both platforms were paired and analysed by 1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et al. Cancer Spearman’s rank-order correlation. This was performed Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. GLOBOCAN 2012, v1.0. to investigate any potential differences between multi- 2. Dukes CE. The classification of cancer of the rectum. 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Assarsson E, Lundberg M, Holmquist G, Bjorkesten J, Bucht Thorsen S, Authors’ contributions Ekman D, et al. Homogenous 96-Plex PEA Immunoassay Exhibiting High (1) Conception and design of the study: MS, ASB, CHR, CD, TS-H, NE, BMS. (2) Sensitivity, Specificity, and Excellent Scalability. PLoS One. 2014;9:e95192. Generation, collection, assembly, analysis and/or interpretation of data: MS, AS B, 13. Hjelm F, Tran B, Fredriksson S: Sensitive detection of cytokines in 1-[mu]l CH R, CD, R E, FS, EG, BE J, KA, RS, MM G, MA, TS-H, NE, BM S, (3) Drafting or serum samples using Proseek[reg]. Nat Meth 2011, 8. revision of the manuscript; MS, AS B, CH R, CD, RE, FS, EG, BE J, KA, MM G, MA, 14. Li D, Chiu H, Gupta V, Chan DW. Validation of a multiplex immunoassay for T S-H, NE, BM S,. (4) Approval of the final version of the manuscript: MS, AS B, serum angiogenic factors as biomarkers for aggressive prostate cancer. CHR, CD, R E, F S, EG, BE J, KA, RS, MM G, MA, TS-H, NE, BM S. All authors read Clin Chim Acta. 2012;413:1506–11. and approved the final manuscript. 15. Nielsen HJ, Brunner N, Jorgensen LN, Olsen J, Rahr HB, Thygesen K, et al. Plasma TIMP-1 and CEA in detection of primary colorectal cancer: a prospective, Acknowledgments population based study of 4509 high-risk individuals. Scand J Gastroenterol. This study was supported with project funding from the Australian National 2011;46:60–9. Health and Medical Research Council (NHMRC APP1010303), NSW Cancer 16. Japink D, Leers MP, Sosef MN, Nap M. CEA in activated macrophages. Council (RG10-04 & RG08-16) and a Macquarie University MQSN grant and New diagnostic possibilities for tumor markers in early colorectal cancer. supported through the Australian School of Advanced Medicine (ASAM), Anticancer Res. 2009;29:3245–51. Mahboob et al. Clinical Proteomics (2015) 12:10 Page 11 of 12

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1542-6416 - (1) Earliest Volume Note Jan. Language of Text Text in: English Refereed Yes Abstracted / Indexed Yes Open Access Yes Serial Type Journal Content Type Academic / Scholarly Format Print Website http://www.clinicalproteomicsjournal.com Email [email protected] Description Provides a forum for novel scientific research in the field of translational proteomics. Subject Classifications Additional Title Details Publisher & Ordering Details Online Availability Abstracting & Indexing Other Availability

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